Investigating the role of Grb2, Dok-3, Cbl and dynein in antigen internalization to provide the second signal for B cell activation
Final Report Abstract
B cells, as part of the adaptive immune response, play a major role in the control and eradication of infections. In response to pathogens such as viruses and bacteria, activation of B cells requires at least two temporally separated signals: (I.) the ligation of the B cell receptor (BCR) with antigen, and (II.) a costimulatory signal, enabling further differentiation of the B cell. Antigen engagement of the BCR leads to the formation of microclusters and the recruitment of an array of intracellular effectors and adaptors which mediate intracellular signalling. Recent data from our lab obtained in the chicken B cell line DT40 demonstrated that two adaptor proteins (Grb2 and Dok-3) as well as the E3 ubiquitin ligase Cbl are recruited to the BCR microcluster after antigen engagement where they mediate recruitment of the minus-end directed microtubule motor dynein. Disruption of dynein expression or function resulted in a severely impaired movement of microclusters towards the center and thus accumulation of antigen. The formation of this central antigen cluster however is a prerequisite for antigen internalisation into endosomes. The endosomal compartment plays a critical role in providing the second signal of B cell activation via TLR9 engagement or loading of antigen on MHC class II molecules. Here, I thus aimed at analysing the role of Grb2, Dok3, Cbl and dynein for antigen gathering, internalization and transport to endosomal compartments in primary splenic B cells to understand the molecular mechanism providing the second signal for B cell activation. By examining the responses of splenic B cells from mice deficient for these mediators in vitro and in vivo I showed here that the mutation in the dynein heavy chains, deficiency in different Cbl isoforms or Dok3 deficiency has only mild effects on antigen internalisation and transport to endosomal compartments. B cells of the different mouse strains studied here demonstrated comparable proliferation and cytokine secretion to endocytosed antigen-CpG conjugates. In addition, antigen processing and loading onto MHC class II molecules, as well as the retrograde transport of these molecules to the surface of the B cells were comparable to wildtype cells. Importantly, B cells deficient for the different mediators were capable to respond to T cell help in vitro and in vivo and demonstrated comparable proliferation. We speculate that this is in part due to redundancy between these molecules in primary splenic B cells. Of note, Dok3 deficiency led to higher in vivo immune responses to influenza virus infection, indicating that Dok3 plays a role in the negative regulation of BCR signalling. Collectively, studying the mutation in the dynein heavy chains, deficiency in different Cbl isoforms or Dok3 deficiency in primary splenic B cells revealed only minor effects of these mediators in providing the second signal for B cell activation.
